ABSTRACT (including UNDERLYING PREMISE and STRATEGY to ENSURE ROBUSTNESS): Our premise is that developing a better method for analyzing proteomes is required to take full advantage of the technological breakthroughs that have occurred in the last 15 years in DNA sequencing. We believe that the difficulties associated with previous proteome analysis methods can be overcome by using an entirely different approach. We propose to test this premise by obtaining affinity reagents to N-terminal amino acids that can be used in a new, massively-parallel method for protein sequencing. Our strategy to ensure a robust, unbiased and reduced risk approach is to develop in parallel the directed evolution of two protein scaffolds and compare them to commercially available and naturally-occurring IgG affinity reagents. We have chosen methods that are extremely well- established in our lab: phage display screening in both bulk and emulsions, phage display library construction, and affinity maturation. The two scaffolds for display include lipocalins and scFv Abs. While not discussed in the proposal, if there is time remaining or if a specific need arises, we may try other scaffolds.